Despite tremendous efforts in molecular, biochemical and cell biological research towards understanding the intra- and extra-cellular mechanisms involved in the transformation of a normal cell into a cancerous one, the number of successful treatments against cancer is few. A major limitation of cancer therapeutics is the problem of delivering pharmacologically relevant compounds, peptidyl mimetics, antisense oligonucleotides, and proteins into cells (Egleton, R. D., and Davis, T. P. (1997) Peptides 18, 1431-1439). Peptide-based drugs have limitations in the form of the poor permeability and selectivity of the cell membrane. These problems are now circumvented by attaching protein translocation domains (PTDs) to the peptides that, can cross the biological membranes efficiently without any dependence on transporters or specific receptors and mediate the intracellular delivery of a range of biological cargos (Schwarze, S. R., Hruska, K. A., and Dowdy, S. F. (2000) Trends Cell Biol. 10, 290-295; Ford, K. G., Souberbielle, B. E., Darling, D., and Farzaneh, F. (2001) Gene Ther. 8, 1-4). The PTD of HIV-1 Tat protein in well-known to mediate transduction of heterologous peptides and biologically active proteins in vitro and in vivo (Ho, A., Schwarze, S. R., Mermelstein, S. J., Waksman, G., and Dowdy, S. F. (2001) Cancer Res. 61, 474-477) and thus has been shown to be of considerable interest for protein therapeutics (Schwarze, S. R., Ho, A., Vocero-Akbani, A., and Dowdy, S. F. (1999) Science 285, 1569-1572).
The integrity of the eukaryotic genome requires several layers of control to ensure that replication of DNA occurs only once during a cell cycle (Elledge, S. J. (1996) Science 274, 1664-1672). For normal cellular functions and tissue homeostasis, accurate transmission of genetic information between generations is required. Dysregulation of the cell cycle control is a hallmark of cancer (Hanahan, D., and Weinberg, R. A. (2000) Cell 100, 57-70). Neoplastic progression has been demonstrated to involve increased genetic instability (Donehower, L. A. (1997) Cancer Surv. 29, 329-352; Harley, C. B., and Sherwood, S. W. (1997) Cancer Surv. 29, 263-284) and there are enough reports revealing that the disruption of multiple pathways is required for the development of cancer (Hunter, T. (1997) Cell 88, 333-346). Inactivation or loss of p53 is a common event associated with the development of approximately 60% of
all human cancers (Levine, A. J. (1997) Cell 88, 323-331; Michael, D., and Oren, M. (2002) Curr. Opin. Genet, Dev. 12, 53-59). The tumor suppressor protein p53 is a short lived, latent transcription factor that is activated and stabilized in response to a wide range of cellular stresses, including DNA damage and activated oncogenes. p53 has been shown to participate in the regulation of several processes, which might inhibit tumor growth, including differentiation, senescence and angiogenesis (Vogelstein, B., Lane, D., and Levine, A. J. (2000) Nature 408, 307-310; Oren, M. (2003) Cell Death, Differ. 10, 431-442). However, central to the function of p53 appears to be the ability to induce both cell cycle arrest and/or apoptosis in stressed cells, partly by activating expression of p53 responsive target genes that mediate these responses (Ashcroft, M., Taya, Y., and Vousden, K. H. (2000) Mol. Cell. Biol. 20, 3224-3233; Woods, D. B., and Vousden, K. H. (2001) Exp. Cell Res. 264, 56-66). Since p53 maintains the genetic stability, it must be under rigorous and complex control. Highly conserved residues in its N- and C-terminal domains are targets for potential post-translational modification via phosphorylation, ubiquitination or acetylation (reviewed by Giaccia and Kastan, 1998: Michael, D. and M. Oren. 2003. Semin. Cancer Biol. 13:49-58). The precise mechanism of p53 activation by cellular stress is of intense interest and may involve both increase in p53 protein level and in the specific activity of p53 by covalent modifications (Harris. S. L., and Levine, A. J. (2005) Oncogene 24, 2899-2908.
The present investigations were aimed at using the protein translocation domain (PTD) of Tat protein to deliver short peptide sequences of tumor suppressor protein SMAR1 both in vitro and in vivo. SMAR1, a recently identified MARBP, was isolated from double positive mouse thymocytes (Chattopadhyay et al., 2000, Genomics). SMAR1, a 68 KDa protein has been previously shown to interact with p53 (Jalota et al., 2005). SMAR1 exists in two alternatively spliced forms: SMAR1L and SMAR1S, with deletion of 39 amino acids in the N-terminus and shares approximately 99% homology with its human homolog, BANP (Birot et al., 2000 Gene 253, 189-196). Interestingly, in numerous cancers, altered expression of several MAR binding proteins have been demonstrated (Liu, W et al., (1999) Cancer Res. 59:5695-5703). In the present work, we show that a 33-mer SMAR1 peptide conjugated to an 11-mer protein transduction domain (PTD) of HIV-1 TAT protein is sufficient enough to inhibit the tumor growth in nude nice. Exposure of cells to this peptide resulted in increased p53 phosphorylation at its serine 15 residue, in turn, activating the p53-mediated cell cycle control. Point-mutation studies of P44 peptide further revealed that the serine 347 residue within the serine-rich motif of SMAR1 plays a pivotal role in mediating the tumor suppressor effect of SMAR1. The 347 serine residue represents the substrate motif for the PKC family of proteins and its phosphorylation is necessary for activating the p53-dependent pathway. We also observe that tumors excised from mice treated with SM mutant peptide showed leaky vascular architecture compared to P44 treated tumors. Interestingly, there was no detectable level of HIF-1α in tumors from mice treated with SMAR1 peptide that is a hallmark of tumor hypoxia. Thus, our results implicate that this SMAR1 peptide can be used, as an alternative drug for cancer therapy.
Major research efforts are aimed at discovery of molecular targets that are specific as well as toxic to cancer cell. Identification of potential targets for therapeutic intervention thus fuels a hope for curing cancer. Peptide-mediated molecular therapeutic delivery systems have currently emerged as an alternative means to effectively substitute or augment present gene therapy technologies, e.g. TAT, VP22, engineered peptides. This invention potentiates the use of P44 peptide of SMAR1 for peptidometic cancer drug design so as to allow therapeutic intervention in the target cell biochemistry without the need to alter its genome.
Inactivation or loss of p53 is a common event associated with the development of approximately 60% of all human cancers. p53 has been shown to participate in the regulation of several processes which might inhibit tumor growth, including differentiation, senescence and angiogenesis. However, central, to the function of p53 appears to be the ability to induce cell cycle arrest and/or apoptosis in stressed cells, at least in part by activating expression of p53-responsive target genes that mediate these responses. Recently, Inventors have identified a novel peptide derived from MAR binding protein; SMAR1 that regulates cell cycle through modulating the activity of p53 and acts as a potent tumor regressor.